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The aim of this study was to provide an efficient tool: reliable, able to increase the molecular diagnosis performance, to facilitate the detection of copy number variants (CNV), to assess genetic risk scores (wGRS) and to offer the opportunity to explore candidate genes. Custom SeqCap EZ libraries, NextSeq500 sequencing and a homemade pipeline enable the analysis of 311 dyslipidemia‐related genes. In the training group (48 DNA from patients with a well‐established molecular diagnosis), this next‐generation sequencing (NGS) workflow showed an analytical sensitivity >99% (n = 532 variants) without any false negative including a partial deletion of one exon. In the prospective group, from 25 DNA from patients without prior molecular analyses, 18 rare variants were identified in the first intention panel genes, allowing the diagnosis of monogenic dyslipidemia in 11 patients. In six other patients, the analysis of minor genes and wGRS determination provided a hypothesis to explain the dyslipidemia. Remaining data from the whole NGS workflow identified four patients with potentially deleterious variants. This NGS process gives a major opportunity to accede to an enhanced understanding of the genetic of dyslipidemia by simultaneous assessment of multiple genetic determinants. [ABSTRACT FROM AUTHOR]

Background: The whitefly Bemisia tabaci is an important agricultural pest with global distribution. This phloem-sap feeder harbors a primary symbiont, "Candidatus Portiera aleyrodidarum", which compensates for the deficient nutritional composition of its food sources, and a variety of secondary symbionts. Interestingly, all of these secondary symbionts are found in co-localization with the primary symbiont within the same bacteriocytes, which should favor the evolution of strong interactions between symbionts. Results: In this paper, we analyzed the genome sequences ofthe primary symbiont Portiera and ofthe secondary symbiont Hamiltonella in the B. tabaci Mediterranean (MED) species in order to gain insight into the metabolic role of each symbiont in the biology of their host. The genome sequences of the uncultured symbionts Portiera and Hamiltonella were obtained from one single bacteriocyte of MED B. tabaci. As already reported, the genome of Portiera is highly reduced (357 kb), but has kept a number of genes encoding most essential amino-acids and carotenoids. On the other hand, Portiera lacks almost all the genes involved in the synthesis of vitamins and cofactors. Moreover, some pathways are incomplete, notably those involved in the synthesis of some essential amino-acids. Interestingly, the genome of Hamiltonella revealed that this secondary symbiont can not only provide vitamins and cofactors, but also complete the missing steps of some of the pathways of Portiera. In addition, some critical amino-acid biosynthetic genes are missing in the two symbiotic genomes, but analysis of whitefly transcriptome suggests that the missing steps may be performed by the whitefly itself or its microbiota. Conclusions: These data suggest that Portiera and Hamiltonella are not only complementary but could also be mutually dependent to provide a full complement of nutrients to their host. Altogether, these results illustrate how functional redundancies can lead to gene losses in the genomes ofthe different symbiotic partners, reinforcing their inter-dependency. [ABSTRACT FROM AUTHOR]

Background: The whitefly Bemisia tabaci is an important agricultural pest with global distribution. This phloem-sap feeder harbors a primary symbiont, “Candidatus Portiera aleyrodidarum”, which compensates for the deficient nutritional composition of its food sources, and a variety of secondary symbionts. Interestingly, all of these secondary symbionts are found in co-localization with the primary symbiont within the same bacteriocytes, which should favor the evolution of strong interactions between symbionts. Results: In this paper, we analyzed the genome sequences of the primary symbiont Portiera and of the secondary symbiont Hamiltonella in the B. tabaci Mediterranean (MED) species in order to gain insight into the metabolic role of each symbiont in the biology of their host. The genome sequences of the uncultured symbionts Portiera and Hamiltonella were obtained from one single bacteriocyte of MED B. tabaci. As already reported, the genome of Portiera is highly reduced (357 kb), but has kept a number of genes encoding most essential amino-acids and carotenoids. On the other hand, Portiera lacks almost all the genes involved in the synthesis of vitamins and cofactors. Moreover, some pathways are incomplete, notably those involved in the synthesis of some essential amino-acids. Interestingly, the genome of Hamiltonella revealed that this secondary symbiont can not only provide vitamins and cofactors, but also complete the missing steps of some of the pathways of Portiera. In addition, some critical amino-acid biosynthetic genes are missing in the two symbiotic genomes, but analysis of whitefly transcriptome suggests that the missing steps may be performed by the whitefly itself or its microbiota. Conclusions: These data suggest that Portiera and Hamiltonella are not only complementary but could also be mutually dependent to provide a full complement of nutrients to their host. Altogether, these results illustrate how functional redundancies can lead to gene losses in the genomes of the different symbiotic partners, reinforcing their inter-dependency. [ABSTRACT FROM AUTHOR]

Bacterial endosymbiosis is an important evolutionary process in insects, which can harbor both obligate and facultative symbionts. The evolution of these symbionts is driven by evolutionary convergence, and they exhibit among the tiniest genomes in prokaryotes. The large host spectrum of facultative symbionts and the high diversity of strategies they use to infect new hosts probably impact the evolution of their genome and explain why they undergo less severe genomic erosion than obligate symbionts. Candidatus Hamiltonella defensa is suitable for the investigation of the genomic evolution of facultative symbionts because the bacteria are engaged in specific relationships in two clades of insects. In aphids, H. defensa is found in several species with an intermediate prevalence and confers protection against parasitoids. In whiteflies, H. defensa is almost fixed in some species of Bemisia tabaci, which suggests an important role of and a transition toward obligate symbiosis. In this study, comparisons of the genome of H. defensa present in two B. tabaci species (Middle East Asia Minor 1 and Mediterranean) and in the aphid Acyrthosiphon pisum revealed that they belong to two distinct clades and underwent specific gene losses. In aphids, it contains highly virulent factors that could allow protection and horizontal transfers. In whiteflies, the genome lost these factors and seems to have a limited ability to acquire genes. However it contains genes that could be involved in the production of essential nutrients, which is consistent with a primordial role for thissymbiont. In conclusion, although both lineages of H. defensa have mutualistic interactions with their hosts, their genomes follow distinct evolutionary trajectories that reflect their phenotype and could have important consequences on their evolvability. [ABSTRACT FROM AUTHOR]

Many insects harbor inherited bacterial endosymbionts. Although some of them are not strictly essential and are considered facultative, they can be a key to host survival under specific environmental conditions, such as parasitoid attacks, climate changes, or insecticide pressures. The whitefly Bemisia tabaci is at the top of the list of organisms inflicting agricultural damage and outbreaks, and changes in its distribution may be associated to global warming. In this work, we have sequenced and analyzed the genome of Cardinium cBtQ1, a facultative bacterial endosymbiont of B. tabaci and propose that it belongs to a new taxonomic family, which also includes Candidatus Amoebophilus asiaticus and Cardinium cEper1, endosymbionts of amoeba and wasps, respectively. Reconstruction of their last common ancestors’ gene contents revealed an initial massive gene loss from the free-living ancestor. This was followed in Cardinium by smaller losses, associated with settlement in arthropods. Some of these losses, affecting cofactor and amino acid biosynthetic encoding genes, took place in Cardinium cBtQ1 after its divergence from the Cardinium cEper1 lineage and were related to its settlement in the whitefly and its endosymbionts. Furthermore, the Cardinium cBtQ1 genome displays a large proportion of transposable elements, which have recently inactivated genes and produced chromosomal rearrangements. The genome also contains a chromosomal duplication and a multicopy plasmid, which harbors several genes putatively associated with gliding motility, as well as two other genes encoding proteins with potential insecticidal activity. As gene amplification is very rare in endosymbionts, an important function of these genes cannot be ruled out. [ABSTRACT FROM AUTHOR]

Background: Sarcoidosis (OMIM 181000) is a multi-systemic granulomatous disorder of unknown origin. Despite multiple genome-wide association (GWAS) studies, no major pathogenic pathways have been identified to date. To find out relevant sarcoidosis predisposing genes, we searched for de novo and recessive mutations in 3 young probands with sarcoidosis and their healthy parents using a whole-exome sequencing (WES) methodology. Methods: From the SARCFAM project based on a national network collecting familial cases of sarcoidosis, we selected three families (trios) in which a child, despite healthy parents, develop the disease before age 15 yr. Each trio was genotyped by WES (Illumina HiSEQ 2500) and we selected the gene variants segregating as 1) new mutations only occurring in affected children and 2) as recessive traits transmitted from each parents. The identified coding variants were compared between the three families. Allelic frequencies and in silico functional results were analyzed using ExAC, SIFT and Polyphenv2 databases. The clinical and genetic studies were registered by the ClinicalTrials.gov - Protocol Registration and Results System (PRS) (https://clinicaltrials.gov) receipt under the reference NCT02829853 and has been approved by the ethical committee (CPP LYON SUD EST – 2 – REF IRB 00009118 – September 21, 2016). Results: We identified 37 genes sharing coding variants occurring either as recessive mutations in at least 2 trios or de novo mutations in one of the three affected children. The genes were classified according to their potential roles in immunity related pathways: 9 to autophagy and intracellular trafficking, 6 to G-proteins regulation, 4 to T-cell activation, 4 to cell cycle and immune synapse, 2 to innate immunity. Ten of the 37 genes were studied in a bibliographic way to evaluate the functional link with sarcoidosis. Conclusions: Whole exome analysis of case-parent trios is useful for the identification of genes predisposing to complex genetic diseases as sarcoidosis. Our data identified 37 genes that could be putatively linked to a pediatric form of sarcoidosis in three trios. Our in-depth focus on 10 of these 37 genes may suggest that the formation of the characteristic lesion in sarcoidosis, granuloma, results from combined deficits in autophagy and intracellular trafficking (ex: Sec16A, AP5B1 and RREB1), G-proteins regulation (ex: OBSCN, CTTND2 and DNAH11), T-cell activation (ex: IDO2, IGSF3), mitosis and/or immune synapse (ex: SPICE1 and KNL1). The significance of these findings needs to be confirmed by functional tests on selected gene variants. [ABSTRACT FROM AUTHOR]

Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy and one of the most common causes of heart failure. TTN-truncating variants represent the most common cause of DCM. Similarly, among other prevalent DCM-causing genes, truncating variants were also frequently detected in BAG3, DSP, FLNC, and LMNA. For these four genes, the current study aims to determine the prevalence of deep intronic pathogenic variants that could lead to splice defects. A next-generation sequencing (NGS) workflow based on whole gene sequencing of BAG3, DSP, FLNC, and LMNA of a cohort of 95 DCM patients, for whom no putatively causative point mutations were identified after NGS of a panel of 48 cardiomyopathy-causing genes, was thus performed. Our approach did not lead us to reconsider the molecular diagnosis of any patient of the cohort. This study suggests that deep splice mutations do not account for a significant proportion of DCM cases. In contrast with MYBPC3 in hypertrophic cardiomyopathy cases, NGS of BAG3, DSP, FLNC, and LMNA whole intronic sequences would not significantly improve the efficiency of molecular diagnosis of DCM probands. [ABSTRACT FROM AUTHOR]

Chromoanagenesis is a cellular mechanism that leads to complex chromosomal rearrangements (CCR) during a single catastrophic event. It may result in loss and/or gain of genetic material and may be responsible for various phenotypes. These rearrangements are usually sporadic. However, some familial cases have been reported. Here, we studied six families in whom an asymptomatic or paucisymptomatic parent transmitted a CCR to its offspring in an unbalanced manner. The rearrangements were characterized by karyotyping, fluorescent in situ hybridization, chromosomal microarray (CMA) and/or whole genome sequencing (WGS) in the carrier parents and offspring. We then hypothesized meiosis‐pairing figures between normal and abnormal parental chromosomes that may have led to the formation of new unbalanced rearrangements through meiotic recombination. Our work indicates that chromoanagenesis might be associated with a normal phenotype and normal fertility, even in males, and that WGS may be the only way to identify these events when there is no imbalance. Subsequently, the CCR can be transmitted to the next generation in an unbalanced and unpredictable manner following meiotic recombination. Thereby, prenatal diagnosis using CMA should be proposed to these families to detect any pathogenic imbalances in the offspring. [ABSTRACT FROM AUTHOR]

Introduction: Depending on the location of insertion of the gained region, F8 duplications can have variable clinical impacts from benign impact to severe haemophilia A phenotype. Aim: To characterize two large Xq28 duplications involving F8 incidentally detected by chromosome microarray analysis (CMA) in two patients presenting severe intellectual disability but no history of bleeding disorder. Methods: Whole genome sequencing (WGS) was performed in order to characterize the two large Xq28 duplications at nucleotide level. Results: In patient 1, a 60–73 kb gained region encompassing the exons 23–26 of F8 and SMIM9 was inserted at the int22h‐2 locus following a non‐homologous recombination between int22h‐1 and int22h‐2. We hypothesized that two independent events, micro‐homology‐mediated break‐induced replication (MMBIR) and break‐induced replication (BIR), could be involved in this rearrangement. In patient 2, the CMA found duplication from 101 to 116‐kb long encompassing the exons 16–26 of F8 and SMIM9. The WGS analysis identified a more complex rearrangement with the presence of three genomic junctions. Due to the multiple micro‐homologies observed at breakpoints, a replication‐based mechanism such as fork stalling and template switching (FoSTeS) was greatly suspected. In both cases, these complex rearrangements preserved an intact copy of the F8. Conclusion: This study highlights the value of WGS to characterize the genomic junction at the nucleotide level and ultimately better describe the molecular mechanisms involved in Xq28 structural variations. It also emphasizes the importance of specifying the structure of the genomic gain in order to improve genotype‐phenotype correlation and genetic counselling. [ABSTRACT FROM AUTHOR]

Background: Structural variants (SVs) include copy number variants (CNVs) and apparently balanced chromosomal rearrangements (ABCRs). Genome sequencing (GS) enables SV detection at base‐pair resolution, but the use of short‐read sequencing is limited by repetitive sequences, and long‐read approaches are not yet validated for diagnosis. Recently, 10X Genomics proposed Chromium, a technology providing linked‐reads to reconstruct long DNA fragments and which could represent a good alternative. No study has compared short‐read to linked‐read technologies to detect SVs in a constitutional diagnostic setting yet. The aim of this work was to determine whether the 10X Genomics technology enables better detection and comprehension of SVs than short‐read WGS. Methods: We included 13 patients carrying various SVs. Whole genome analyses were performed using paired‐end HiSeq X sequencing with (linked‐read strategy) or without (short‐read strategy) Chromium library preparation. Two different bioinformatic pipelines were used: Variants are called using BreakDancer for short‐read strategy and LongRanger for long‐read strategy. Variant interpretations were first blinded. Results: The short‐read strategy allowed diagnosis of known SV in 10/13 patients. After unblinding, the linked‐read strategy identified 10/13 SVs, including one (patient 7) missed by the short‐read strategy. Conclusion: In conclusion, regarding the results of this study, 10X Genomics solution did not improve the detection and characterization of SV. [ABSTRACT FROM AUTHOR]

TCF4 gene (18q21.1) encodes for a transcription factor with multiple isoforms playing a critical role during neurodevelopment. Molecular alterations of this gene are associated with Pitt-Hopkins syndrome, a severe condition characterized by intellectual disability, specific facial features and autonomic nervous system dysfunction. We report here three patients presenting with structural variations of the proximal part of TCF4 associated with a mild phenotype. The first patient is a six-years-old girl carrier of a pericentric inversion of chromosome 18, 46,XX,inv(18)(p11.2q21.1). Whole genome sequencing (WGS) characterized the breakpoint at the base-pair level at chr18:1262334_1262336 and chr18:53254747_53254751 (hg19). This latter breakpoint disrupted the proximal promotor region of TCF4 in the first intron of the gene. The second and third patients are a son and his mother, carrier of a 46 kb deletion characterized by high-resolution chromosomal micro-array and WGS (chr:18:53243454_53287927, hg19) encompassing the first three exon of TCF4 gene and including the proximal promotor region. Expression studies on blood lymphocytes in these patients showed a marked decrease of mRNA level for long isoforms of TCF4 and an increased level for shorter isoforms. The patients described here, together with previously reported patients with proximal structural alterations of TCF4, help to delineate a phenotype of mild ID with non-specific facial dysmorphism without characteristic features of PTHS. It also suggests a gradient of phenotypic severity inversely correlated with the number of intact TCF4 promotor regions, with expression of short isoforms compensating in part the loss of longer isoforms. [ABSTRACT FROM AUTHOR]

Chromoanagenesis represents an extreme form of genomic rearrangements involving multiple breaks occurring on a single or multiple chromosomes. It has been recently described in both acquired and rare constitutional genetic disorders. Constitutional chromoanagenesis events could lead to abnormal phenotypes including developmental delay and congenital anomalies, and have also been implicated in some specific syndromic disorders. We report the case of a girl presenting with growth retardation, hypotonia, microcephaly, dysmorphic features, coloboma, and hypoplastic corpus callosum. Karyotype showed a de novo structurally abnormal chromosome 14q31qter region. Molecular characterization using SNP-array revealed a complex unbalanced rearrangement in 14q31.1-q32.2, on the paternal chromosome 14, including thirteen interstitial deletions ranging from 33 kb to 1.56 Mb in size, with a total of 4.1 Mb in size, thus suggesting that a single event like chromoanagenesis occurred. To our knowledge, this is one of the first case of 14q distal deletion due to a germline chromoanagenesis. Genome sequencing allowed the characterization of 50 breakpoints, leading to interruption of 10 genes including YY1 which fit with the patient's phenotype. This precise genotyping of breaking junction allowed better definition of genotype-phenotype correlations. [ABSTRACT FROM AUTHOR]

Williams Beuren syndrome (WBS) is a multiple malformations/intellectual disability (ID) syndrome caused by 7q11.23 microdeletion and clinically characterized by a typical neurocognitive profile including excessive talkativeness and social disinhibition, often defined as "overfriendliness" and "hyersociability". WBS is generally considered as the polar opposite phenotype to Autism Spectrum Disorder (ASD). Surprisingly, the prevalence of ASD has been reported to be significantly higher in WBS (12%) than in general population (1%). Our study aims to investigate the molecular basis of the peculiar association of ASD and WBS. We performed chromosomal microarray analysis and whole exome sequencing in six patients presenting with WBS and ASD, in order to evaluate the possible presence of chromosomal or gene variants considered as pathogenic.Our study shows that the presence of ASD in the recruited WBS patients is due to i) neither atypically large deletions; ii) nor the presence of pathogenic variants in genes localized in the non-deleted 7q11.23 allele which would unmask recessive conditions; iii) moreover, we did not identify a second, indisputable independent genetic diagnosis, related to pathogenic Copy Number Variations or rare pathogenic exonic variants in known ID/ASD causing genes, although several variants of unknown significance were found. Finally, imprinting effect does not appear to be the only cause of autism in WBS patients, since the deletions occurred in alleles of both maternal and paternal origin.The social disinhibition observed in WBS does not follow common social norms and symptoms overlapping with ASD, such as restricted interests and repetitive behavior, can be observed in "typical" WBS patients: therefore, the terms "overfriendliness" and "hypersociability" appear to be a misleading oversimplification.The etiology of ASD in WBS is likely to be heterogeneous. Further studies on large series of patients are needed to clarify the observed variability in WBS social communication, ranging from excessive talkativeness and social disinhibition to absence of verbal language and social deficit. [ABSTRACT FROM AUTHOR]